Upon infection by a pathogen, the host's immune system recognizes antigens on or in the pathogen and directs an immune response against the antigen-containing pathogen. During this response, there is an increase in the number of immune cells specific to the antigens of the pathogen and some of these cells remain after the infection subsides. The presence of the remaining cells prevents the pathogen from establishing infection when the host is subjected to the pathogen at a later time. This is referred to as protective immunity.
Vaccines provide protective immunity against pathogens by presenting a pathogen's antigens to the immune system without causing disease. Several methods have been developed to allow presentation of antigens without disease-causing infection by the pathogen. These include using a live but attenuated pathogen, an inactivated pathogen, or a fragment (subunit) of the pathogen.
Because therapy for many viral infections remains elusive, it is preferred to prevent or moderate infection through vaccination rather than treat the infection after it occurs. Examples of particularly problematic infectious viruses are those of the orthomyxoviridae, especially influenza virus, paramyxoviridae, flaviviridae, togaviridae, rhabdoviridae, and coronaviridae families. Millions of people are vaccinated against one or more members of these virus families each year.
While some viruses will propagate well in cell culture, others require propagation in embryonated chicken eggs with virus recovery from allantoic fluids. Influenza vaccine has been supplied to the populace for many years as a multi-strain combination product recovered from the allantoic fluids of embryonated chicken eggs. Three strains, selected annually from a large panel of strains, are grown, purified, and pooled to create a given vaccine. The growth of each selected strain of influenza can vary markedly, often leading to difficulties in efficiently meeting the annual market demand for such a trivalent vaccine.
Various methods have been proposed to improve and/or simplify the recovery of virus or viral products from feedstock. U.S. Pat. No. 3,627,873 describes a process in which virus is extracted from concentrated allantoic fluid feedstock using diethyl ether and methylacetate. Still further yield improvements are said to have been obtained using multiple extractions with both butyl and ethylacetates according to U.S. Pat. No. 4,000,257.
U.S. Pat. No. 3,316,153 describes a multi-step extraction process, aimed at separating virus particles from cellular debris and is assertedly applicable to feedstocks that derive from virus-infected chick allantoic fluid or from cell or tissue-culture fluids. In this method, virus adsorbed to precipitated calcium phosphate is dispersed in EDTA at pH 7.8-8.3, causing dissociation and an EDTA-based sequestering of the soluble calcium, thereby releasing the virus for recovery. The resulting virus-containing solution is dialyzed against water or preferably an aqueous glycine-sodium chloride solution to reduce the EDTA and phosphate content.
U.S. Pat. No. 4,724,210 describes methods for purification of influenza using ion exchange chromatography. An influenza-containing solution, e.g. allantoic fluid, is passed through cellulose sulfate column wherein the virus is adhered to the column packing. The column is subsequently washed and virus eluted with a solution containing 1.0 M to 1.5 M sodium chloride. This is followed by a 4.99 M sodium chloride wash.
In WO 02/067983, preparation of a split influenza vaccine is described as involving moderate-speed centrifugation to clarify allantoic fluid, adsorption of the clarified fluid on a CaHPO4 gel, followed by resolublization with an EDTA-Na2 solution. See also WO 02/08749 describing the same process.
In U.S. Pat. No. 4,327,182, allantoic fluid feedstocks from the growth of influenza virus are subjected to a multi-stage extraction process aimed at recovering influenza subunits, haemagglutinin (HA) and neuraminidase (NA). The technique relies on a concentration step in which virus feedstock is present with detergent and a saline solution followed by successive filtration to remove non-viral particles.
U.S. Pat. No. 3,962,421 describes a method for the disruption of influenza viruses. Allantoic fluid is subjected to high-speed centrifugation. The resulting pellet is resuspended in saline and ball-milled for 12-15 hours to create a virus suspension. The virus suspension is then treated with phosphate-ester to disrupt the virus particles into lipid-free particles (subunits) that carry the surface antigens of intact viruses.
In U.S. Pat. No. 3,874,999, allantoic fluids containing influenza virus are centrifuged at low speeds to remove gross particles. The virus is then removed from the supernatant by high-speed centrifugation and resuspended in a phosphate buffer. Nonvirus proteins and lipids are removed by treatment of the suspension with 0.1-0.4 M magnesium sulfate at an alkaline pH for 16-18 hours at 4° C. The resultant suspension is clarified by low speed centrifugation and the virus is purified from the resulting supernatant.
Of particular interest to the background of the invention are viral recovery manipulations involving the contact of non-allantoic fluid virus sources with solutions having elevated concentrations of one or more salts and studies of the effect of various salt concentrations on purified virus.
Some processes assertedly provide for increased yields or greater purity of virus when infected cells are contacted or incubated with solution containing elevated salt concentrations followed by purification of the virus from the solution.
In WO 99/07834, herpesvirus infected Vero cell cultures are incubated in a hypertonic aqueous salt solution (e.g., 0.8 to 0.9 M NaCl) for several hours. The solution is then removed and herpesvirus harvested from the solution. This method was asserted to be superior to methods wherein the cells are subjected to ultrasonic disruption.
Others have addressed contacting virus-infected cultured cells with elevated salt concentrations.
U.S. Pat. No. 5,506,129 reports increased yields of hepatitis A virus after growing infected BS-C-1 cells in growth medium containing ˜0.3 M NaCl.
Karakuyumchan et al. (Acta virol.:155-158, 1981) reports that rabies virus obtained after shaking infected brain tissue in a 0.3 M NaCl containing buffered solution lacks neuroallergenic activity caused by residual brain tissue.
Pauli and Ludwig (Virus Research, 2:29-33, 1985) reports increased yields of Boma disease virus from a virus-infected cell lines grown in medium containing ˜0.3 M NaCl.
Various groups have studied the effect of contacting purified viruses with elevated salt concentrations on the characteristics of the virus.
In Breschkin et al. (Virology, 80:441-444, 1977), a particular mutated measles virus lacking hemagglutination activity in isotonic saline has wild-type level hemagglutination activity in 0.8 M (NH4)2SO4, whereas the high salt has no effect on the hemagglutination activity of a wild-type virus.
Wallis and Melnick (Virology, 16:504-506, 1962) report that, while high salt (1 M MgCl2, 1 M CaCl2, or 2 M NaCl) prevents heat inactivation of polio, coxsackie, and ECHO viruses, 1 M MgCl2 enhances inactivation of adeno-, papova-, herpes-, myxo-, arbor, and poxviruses.
In Willkommen et al. (Acta virol., 27:407-411, 1983), purified lyophilized influenza virus is reconstituted in buffered saline containing increasing concentrations of NaCl (up to 1.15 M). Subsequently, the reconstituted virus is cleaved with detergent and a single-radial-immunodiffusion (SRD) test performed. With some strains of influenza virus, increasing the salt concentration in the reconstitution buffer shows no effect on the results of an SRD test to hemaggluinin (HA). However, other strains, when reconstituted in buffered saline containing 1.15 M NaCl, give a HA concentration in the SRD test that is twice that of the same strain reconstituted in buffered saline containing 0.15 M NaCl. The authors identify viral aggregation as possibly blocking detergent penetration and attenuated the SRD response.
Molodkina et al. (Colloids and Surfaces A: Physicochemical and Engineering Aspects, 98:1-9, 1995) report that increasing salt concentrations up to 0.3 M NaCl leads to dispersion of purified influenza virus aggregates.
Sudnik et al. (Vyestsi Akademii Navuk BSSR Syeryya Biyalahichnykh Navuk, 6:71-77, 1985) report high ionicity can partially offset the destruction of the influenza virus envelope at pH 2.2.
Also of interest to the background of the invention are the results of studies by Makhov et al. (Voprosy Virusologii, 34(2):274-279, 1989) on the proportion of filamentous influenza virions in allantoic fluids. In a context divorced from virus recovery, Makhov et al. report that presence of filamentous influenza virions in allantoic fluids is strain specific and ionic-strength dependent. Allantoic fluids were examined using electron microscopy to determine the presence of filamentous virions. The occurrence of filamentous virions in the allantoic fluid for one particular influenza strain was 7.1%. When the NaCl concentration was raised to 0.25 M or 1.0 M, the occurrence was reduced to 0.37% and 0.16%, respectively.
Thus, there remains a need in the art for an improvement of the purity and yield of viruses from allantoic fluid of virus-infected chick embryos.